YY2/BUB3 Axis promotes SAC Hyperactivation and Inhibits Colorectal Cancer Progression via Regulating Chromosomal Instability

Abstract Spindle assembly checkpoint (SAC) is a crucial safeguard mechanism of mitosis fidelity that ensures equal division of duplicated chromosomes to the two progeny cells. Impaired SAC can lead to chromosomal instability (CIN), a well‐recognized hallmark of cancer that facilitates tumor progression; paradoxically, high CIN levels are associated with better therapeutic response and prognosis. However, the mechanism by which CIN determines tumor cell survival and therapeutic response remains poorly understood. Here, using a cross‐omics approach, YY2 is identified as a mitotic regulator that promotes SAC activity by activating the transcription of budding uninhibited by benzimidazole 3 (BUB3), a component of SAC. While both conditions induce CIN, a defect in YY2/SAC activity enhances mitosis and tumor growth. Meanwhile, hyperactivation of SAC mediated by YY2/BUB3 triggers a delay in mitosis and suppresses growth. Furthermore, it is revealed that YY2/BUB3‐mediated excessive CIN causes higher cell death rates and drug sensitivity, whereas residual tumor cells that survived DNA damage‐based therapy have moderate CIN and increased drug resistance. These results provide insights into the role of SAC activity and CIN levels in influencing tumor cell survival and drug response, as well as suggest a novel anti‐tumor therapeutic strategy that combines SAC activity modulators and DNA‐damage agents.


Introduction
Chromosomal instability (CIN) is characterized by an increased rate of chromosomal structural and numerical abnormalities due to aberrant segregation.Chromosomal abnormalities cause significant fitness costs to normal cells, leading to various disorders, including metabolic alterations, proteotoxic stress, cell cycle arrest, and senescence. [1]urthermore, it is the most common cause of spontaneous abortion and severe developmental defects. [2]Despite these deleterious effects, CIN is a well-recognized cancer hallmark, with ≈90% of tumors displaying complex karyotypes, including structural and numerical CIN. [3]11] Impaired activity of the spindle assembly checkpoint (SAC), one of the major cell cycle checkpoints, is a major cause of CIN. [12]As a checkpoint at the metaphase-anaphase transition point, it ensures that sister chromatids are segregated equally into the two progeny cells and is thus the last checkpoint to guarantee mitotic fidelity through the passage of correct, intact genetic information to the progeny cells. [13]SAC activity prevents premature cohesion cleavage, sister chromatid segregation, and mitotic exit by suppressing securin and cyclin B ubiquitination/proteasomal degradation before all sister chromatids are lined up at the equator and attached to spindle microtubules. [13]efect in SAC triggers premature sister chromatid segregation and mitotic exit, leading to increased CIN and faster mitosis, thereby serving as a driving force for tumorigenesis. [14,15]Similarly, SAC hyperactivation also induces CIN; however, it leads to mitotic delay and tumor suppression. [16,17]The role of SAC hyperactivation in tumorigenesis remains poorly understood.Furthermore, the reasons underlying the different outcomes related to SAC defects and hyperactivation remain to be explored.
Although mutations in SAC genes are rare, alterations in SAC gene expression are frequently found in tumor cells, suggesting a crucial role for transcriptional regulation in aberrant SAC activity in tumor cells. [18,19]However, the mechanisms underlying SAC transcriptional regulation have not yet been fully elucidated.In this study, we utilized a cross-omics approach to search for potential transcriptional regulators of SAC genes and identified yin yang 2 (YY2) as a novel budding uninhibited by benzimidazole 3 (BUB3) transcriptional regulator.Our results showed that YY2/BUB3 axis positively modulates SAC activity, prolongs mitotic time, and suppresses colorectal cancer (CRC) cells survival.However, despite of their opposite effects on SAC activity and CRC cells survival, both YY2/BUB3 deficiency and overexpression lead to increased CIN.We further revealed that different CIN degrees induced by YY2/BUB3 alterations is crucial for determining CRC cell survival and drug response, notably, excessive CIN level leads to cell death while moderate CIN is beneficial for CRC cells.Finally, our results showed a strong correlation between moderate CIN and drug resistance, such as that found in residual tumor cells resistant to DNA damage-inducing agents and/or SAC activation.In contrast, the induction of excessive CIN in those cells by YY2 overexpression sensitizes them to DNA damage-inducing agents, suggesting a novel anti-tumor therapeutic approach by combining SAC activator and DNA damageinducing agents.

Identification of Transcriptional Regulators Modulating SAC
To identify a novel modulator of the SAC and subsequently tumor cell CIN, we screened genes involved in both "mitotic sister chromatid segregation" and "mitotic spindle assembly checkpoint signaling" from the Gene Ontology (GO) database (QuickGO, http://www.ebi.ac.uk/QuickGO;GO numbers: 0000070 and 0007094; respectively) and obtained 195 genes.After excluding non-human-origin and hypothetical genes, we further screened genes that have been reviewed and annotated using the manual-curation process in UniProtKB and referenced based on a PubMed Unique Identifier (PMID).The 20 genes identified in this screening were subjected to transcription factor (TF) enrichment analysis using enrichment analysis version 3 (ChEA3, https://maayanlab.cloud/chea3/), a web server application developed to conduct TF enrichment analysis utilizing gene set libraries from published omics assay data extracted from multiple sources [20] (Figure 1A).Among the top 17 TFs obtained, eight TFs were known SAC modulators, thus confirming the validity of this screening method; [21][22][23][24] whereas nine TFs were potential novel SAC modulators (Figure 1B).
TFs that regulate the cell cycle usually exhibit oscillating levels during cell cycle progression. [25]Thus, we first determined the timing of each cell cycle phase by synchronizing HCT116 CRC cells in the G 0 /G 1 phase using serum starvation (Figure S1A, Supporting Information).The percentage of cells in the G 0 /G 1 phase decreased from 78.12% immediately after serum starvation release to 14.11% 16 h later, whereas cells in the S phase increased from 10.20% to a peak at 76.89% after 16 h, a timing necessary for HCT116 cells to progress into S phase after starvationinduced G0 phase by serum starvation as reported by previous study. [26]Meanwhile, cells in the G 2 /M phase reached 55.69% at 24 h after serum starvation release.Cell cycle-dependent expression analysis of the nine novel SAC modulators revealed that significant expression of ZFP69, ZFP69B, ZNF93, ZNF730, and ZNF878 could not be detected in any cell cycle state, while those of ZNF215, ZNF280C, and ZNF888 did not show significant differences during cell cycle progression.Meanwhile, YY2 mRNA expression clearly increased during cell cycle progression and peaked during the M phase (Figure 1C).A more detailed timecourse investigation showed that YY2 mRNA expression peaked at 20 h (Figure S1B, Supporting Information), whereas its protein level started to increase significantly at 16 h and peaked at 24 h after serum starvation release (Figure 1D).Meanwhile, the levels of G 1 and G 2 /M phase cyclins, specifically cyclins D and B, peaked at 4 h and 24 h after serum starvation release, respectively, further confirming the relationship between YY2 expression and the M phase.
SAC delays mitotic exit by stabilizing securin and suppressing APC/C activity.To further explore the role of YY2 in regulating SAC activity and subsequently mitotic exit, we altered YY2 expression in HCT116 cells, a near-diploid human CRC cell line with a mitotic time similar to that of normal cells and considered CIN-negative cells (Figure S1C,D, Supporting Information). [27]s shown by time-lapse microscopic images, YY2 overexpression delayed the mitosis, whereas YY2 knockout significantly accelerated it (Figure 1E,F, Videos S1-S4, Supporting Information).Measurement of mitotic time revealed that YY2 overexpression prolonged the mitotic time from 53.6 min to 73.7 min, while YY2 knockout significantly shortened it from 52.8 min to 42.8 min (Figure 1G,H).Notably, when YY2 was reintroduced into YY2knocked out cells to the level similar to control, the mitotic time was also restored to wild-type level, further confirming the function of YY2 in regulating mitosis (Figure S1E-G, Supporting Information).Next, we modulated YY2 expression and synchronized cells in the M phase using nocodazole, a metaphasearresting SAC activator.YY2 overexpression significantly slowed down the degradation of securin and cyclin B, an event that marks mitotic exit (Figure S2A,B, Supporting Information), whereas knocking out YY2 accelerated their degradation (Figure S2C,D, Supporting Information).Moreover, to further characterize the role of YY2 in regulating SAC activity, we analyzed the mitotic time of nocodazole-treated cells.Compared to nocodazole-treated control cells, YY2-overexpressed cells exhibited prolonged mitotic time under nocodazole treatment (Figure S2E, Supporting Information), while YY2-knocked out cells underwent shorter mitotic arrest (Figure S2F, Supporting Information).Furthermore, to confirm that the observed prolonged mitosis in YY2overexpressed cells was due to increase in SAC strength rather than mere activation of SAC, we investigated the ability of YY2overexpressed cells treated with nocodazole to sustain an extended mitotic arrest. [28]YY2 overexpression led to stronger SAC activity, as demonstrated by fewer cells that exit mitosis under constant SAC activation (Figure 1I); meanwhile, YY2 knockout exerted the opposite (Figure 1J).These results solidified the role of YY2 in promoting SAC activity.Together, our results revealed that YY2, of which the expression oscillates during the cell cycle and reaches its peak in the M phase, is a novel SAC modulator that controls mitotic progression.

YY2-Mediated SAC Regulation is Crucial for its Tumor Suppressive Effect
[31][32] A comparative analysis using clinical CRC tissues and corresponding normal adjacent tissues revealed a significant decrease in YY2 mRNA and protein levels in CRC tissues (Figure S3A,B, Supporting Information).Furthermore, YY2 overexpression significantly suppressed the viability and colony-formation potential of HCT116 cells (Figure S3C,D, Supporting Information).As a decrease in cell viability might be due to a decrease in proliferation, increase in cell death, or both, we next examined the effect of YY2 on HCT116 cell proliferation and cell death.YY2 overexpression robustly suppressed CRC cell proliferation, as indicated by the decrease in EdU-positive cells (Figure S3E, Supporting Information), while increasing the cell death rate (Figure 2A), suggesting that YY2 may exert its tumor suppressive function most plausibly by decreasing cell proliferation and promoting cell death.In contrast, the viability, colony-formation potential, and proliferation potential of HCT116 YY2KO cells were significantly higher than those of wild-type cells (Figure S3F-H, Supporting Information).Interestingly, YY2 knockout did not significantly affect the cell death rate (Figure 2B), most plausibly because of the overall low cell death rate.Notably, the cell death rate increased significantly when YY2 was reintroduced into YY2-knocked out cells in excessive level, further confirming that YY2 overexpression could induce cell death (Figure S3I, Supporting Information).It is note-worthy that proper level SAC activity is important to prevent event that can induce cell death, such as chromosome missegregation.
To examine whether YY2 exerts its tumor suppressive effect by regulating SAC activity, we treated YY2-overexpressed HCT116 cells with reversine, a SAC activity inhibitor. [33,34]In control cells, reversine treatment reduced cyclin B and securin accumulation to levels significantly lower than those in control cells; meanwhile, in YY2-overexpressed cells, it reduced cyclin B and securin accumulation induced by YY2 overexpression to levels near to those in control cells (Figure S3J, Supporting Information).Furthermore, reversine treatment re-suppressed mitotic time prolonged by YY2 overexpression from 72.7 min to 52.9 min, which was also similar to control (Figure 2C,D, Video S5, Supporting Information).These results indicated that reversine treatment restored YY2-induced SAC hyperactivation to a level similar to control.Accordingly, while reversine treatment suppressed the proliferation, viability, and colony-formation potential of control HCT116 cells, it restored these potentials in YY2-overexpressed HCT116 cells (Figure 2E; Figure S3K,L, Supporting Information).Similarly, reversine treatment increased cell death rate in control HCT116 cells, while suppressing that of YY2 overexpressed cells (Figure 2F).These results not only reveal that proper level of SAC activity is crucial to support cell survival but also YY2-mediated SAC activation and mitotic regulation are crucial for exerting its tumor suppressive effect.

YY2 Regulates SAC by Directly Activating BUB3 Transcription
To analyze the molecular mechanism underlying YY2-mediated regulation of SAC activity, we performed a cross-omics analysis to identify its potential transcriptional target.To this end, RNAsequencing (RNA-seq) data obtained in our previous study (https: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184138) using YY2-overexpressed HCT116 cells [29] were analyzed for differentially expressed genes (DEGs; Figure 3A).The DEGs (fold-change > 1.01; P < 0.05) were then analyzed for enriched GO terms and Reactome pathways using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/).GO analysis indicated that YY2 overexpression enriched genes involved in cell division, mitotic nuclear division, sister chromatid cohesion, and negative regulation of ubiquitin-protein ligase activity in mitosis (Figure 3B); meanwhile, Reactome analysis revealed the enrichment of APC/C-Cdc20-mediated degradation of securin, separation of sister chromatids, mitotic prometaphase, and resolution of sister chromatid cohesion (Figure 3C), indicating a possible prominent role of YY2 in regulating sister chromatid segregation.) mRNA expression levels of potential novel SAC modulators at each cycle phase, as examined using qRT-PCR.D) YY2, cyclin D, and cyclin B protein expression levels in HCT116 cells at indicated timepoints after serum starvation release, as determined by western blotting.E-F) Mitotic time of YY2-overexpressed HCT116 cells (E) and HCT116 YY2KO cells (F), as determined using time-lapse microscopy.Representative images (scale bars: 20 μm) are shown.G-H) Scatter plots showing the timelength from nuclear envelope breakdown (NEBD) to anaphase of YY2-overexpressed HCT116 cells (G) and HCT116 YY2KO cells (H) (total n = 60, pooled from three independent experiments).I,J) Time-lapse analysis of duration of mitosis in YY2-overexpressed HCT116 cells (I) and HCT116 YY2KO cells (J) arrested with nocodazole (final concentration: 100 ng mL −1 ; total n = 150, pooled from three independent experiments).Cells transfected with pcCon, wild-type HCT116, or unsynchronized cells were used as controls.-actin was used for qRT-PCR normalization and as western blotting loading control.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way analysis of variance (ANOVA).pcCon: pcEF9-Puro; Unsync: unsynchronized; **P < 0.01; NS: not significant; ND: not detected.
To identify potential YY2 transcriptional targets that regulate SAC activity, we overlapped DEGs identified by RNA-seq with those identified by chromatin immunoprecipitation (ChIP)sequencing using an anti-YY2 antibody reported previously, [30] as well as genes obtained from QuickGO screening (Figure 1A).Two genes, BUB3 and TEX14, were identified as potential YY2 direct transcriptional targets (Figure 3D).BUB3 is a component of the SAC complex that regulates sister chromatid segregation and mitotic progression; [13] meanwhile, TEX14 is required for the formation of intercellular bridges during meiosis. [35]In accordance with previous studies, [35,36] analysis of the TEX14 expression profiles using GTEx v8 expression data across 29 tissues revealed its testis specificity (Figure S4A, Supporting Information).Furthermore, absolute qRT-PCR results showed that the copy number of TEX14 was very low in HCT116 cells (Figure S4B, Supporting Information).Meanwhile, BUB3 mRNA increased nearly twofold upon YY2 overexpression (Figure S4C, Supporting Information), whereas its protein level was positively correlated with YY2 in YY2-knock-down and YY2-overexpressed HCT116 cells (Figure 3E).Moreover, to avoid the effect of YY2 oscillation during cell cycle, we arrested the HCT116 cells at G 0 /G 1 phase using serum starvation and investigated the effect of altering YY2 expression on BUB3.The results consistently demonstrated that YY2 alteration positively correlates with BUB3 expression level, further confirming the direct regulation of YY2 on BUB3 expression (Figure S4D,E, Supporting Information).
To elucidate the role of BUB3 in YY2-induced SAC activity and prolonged mitosis, we first analyzed the effect of BUB3 overexpression on mitotic time.The level of BUB3 in cells transfected with BUB3 overexpression vectors was confirmed (Figure S4F,G, Supporting Information).Similar to that of YY2 overexpression, BUB3 overexpression in HCT116 cells prolonged the mitotic time from 51.3 min to 80.5 min (Figure S4H,I and Video S6, Supporting Information) and slowed the degradation rates of cyclin B and securin proteins (Figure S4J-L, Supporting Information).BUB3 overexpression restored the length of HCT116 cells mitotic time, which was shortened to 43.3 min by YY2 knockout, to a level similar to that observed in the control (50 min and 52.9 min, respectively; Figure S5A-C and Video S7, Supporting Information).Next, we constructed two shRNA expression vectors targeting different sites of BUB3 and selected shBUB3-2 (refers as shBUB3 hereafter), which had a higher suppressive effect, for further experiments (Figure S5D,E, Supporting Information).Similar to the trend observed after knocking out YY2, knocking down BUB3 shortened the mitotic time of HCT116 cells from 52.7 min to 40.5 min and resuppressed the mitotic time, which was prolonged by YY2 overexpression, from 71.4 min to 46.8 min (Figure S5F-H and Videos S8,S9, Supporting Information).Accordingly, BUB3 knockdown restored HCT116 cell viability and cell death, which was suppressed by YY2 overexpression (Figure S5I,J, Supporting Information).These results confirmed that YY2 regulates SAC and subsequently, mitotic progression and tumorigenic potential by enhancing BUB3 expression.

Both YY2 Knockout and Overexpression induce CIN
Defects in SAC activity disrupt the mechanism that guarantees the proper arrangement of chromosomes at the equator and attachment to the mitotic spindle, making them prone to missegregation, and therefore, is a well-known causal factor of CIN induction.Thus, we examined the effect of YY2 silencing on CIN indicators.We observed significant increases in the frequency of cells with DNA content > 4N (Figure 4A) as well as in nuclear size (Figure 4B) in HCT116 YY2KO cells, indicating the increase of the frequency of polyploid cells.Metaphase spread analysis results showed a significant increase in the karyotypic heterogeneity of HCT116 YY2KO cells, as nearly 80% of HCT116 cells had 45-47 chromosomes and fewer than 4% had more than 47 chromosomes, whereas only approximately 50% of HCT116 YY2KO cells had 45-47 chromosomes and the percentage of cells with more than 47 chromosomes increased to nearly 35% (Figure 4C).Single karyotype analysis further confirmed the increase of karyotypic heterogeneity in HCT116 YY2KO , indicated by clones with gain or loss of chromosome (Figure 4D; Figure S6A, Supporting Information).
Failed sister chromatid segregation leads to the formation of partly unsegregated sister chromatids or chromosome bridges, of which DNA fragments are subsequently wrapped by a nuclear membrane-like structure during cytokinesis to form a micronucleus, a small, nucleus-like structure in the cytoplasm.YY2 knockout significantly increased the chromosome bridge frequency (Figure 4E) and concomitantly increased the micronucleus rate (Figure 4F), which could lead to the formation of aneuploid cell.Meanwhile, BUB3 overexpression could partially abrogate the frequencies of polyploid cells and chromosome bridges, as well as the micronucleus rate in HCT116 YY2KO cells, while reintroduction of YY2 into YY2-knocked out cells fully restored them (Figure S6B-D, Supporting Information).Together, these results suggest that defects in the YY2/BUB3 pathway could contribute to CIN induction in tumor cells, most plausibly due to impaired SAC activity.Surprisingly, YY2 overexpression, which induced SAC hyperactivation and mitotic delay, also increased the number of polyploid cells, nuclear size, karyotype heterogeneity, chromosome bridge frequency, and micronucleus rates (Figure 4G-L; Figure S6E, Supporting Information).These results suggest that YY2 overexpression might also increase CIN.
Given that BUB3 is a component of SAC, we overexpressed BUB3 and examined its effect on CIN.BUB3 overexpression induced cell death (Figure S7A, Supporting Information) and increased CIN phenotypes (Figure S7B-E, Supporting Information).These results conform with previous studies showing that SAC activation could also induce CIN and furthermore, cell death. [16,39]To further confirm the causal relation between YY2 overexpression-induced SAC hyperactivation and CIN, we suppressed SAC activity in HCT116 cells overexpressing YY2.Suppression of BUB3 or treatment with reversine significantly decreased the number of polyploid cells (Figure S8A,B, Supporting Information), chromosome bridge frequency (Figure S8C,D, Supporting Information), and micronucleus rate (Figure S8E,F, Supporting Information) in HCT116 cells overexpressing YY2, which is likely due to the suppression of SAC activity to near wild-type level in these cells as shown in Figure S5F,G (Supporting Information) Together, our results clearly show that both YY2 knockout and overexpression induced CIN.

CIN Levels Determine Tumor Cell Survival
The aforementioned results showing that both YY2 knockout and overexpression induced CIN were intriguing, as YY2 knockout suppressed SAC activity, shortened the mitotic time, and promoted HCT116 cell proliferation, whereas YY2 overexpression induced SAC hyperactivation, prolonged the mitotic time, enhanced cell death, and suppressed HCT116 cell proliferation.[11] Using TCGA CRC data set, we next performed correlation analysis between relapse-free survival rate of CRC patients treated with oxaliplatin, a drug currently used as first-line therapy for treating CRC in clinical practice, [40] and the pretreatment levels of CIN70, a 70-gene signature that has been established as surrogate of CIN. [41]The results showed that the moderate CIN levels, i.e., the second and third quartiles, were associated with poor prognosis; meanwhile, high levels of CIN, i.e., the fourth quartile, were associated with better survival, suggesting that excessive CIN might be deleterious for tumors (Figure 5A).
We next examined the survival of YY2-overexpressed cells.To control the timing of YY2 overexpression, we established a Tet-On YY2 overexpression system (Figure S9A,B, Supporting Information).The cell death rate increased in a time-dependent manner after the induction of YY2 overexpression (Figure 5B); while treatment with reversine cancelled it (Figure 5C), suggesting the correlation between YY2 overexpression-induced SAC hyperactivation and cell death.Given that YY2 overexpression also led to increased CIN (Figure 4G-L), these results indicated the possibility that YY2-induced cell death might be correlated with SAC hyperactivation-induced CIN.However, unlike the cell death rate, the micronucleus rate started to decline after reaching its peak at 48 h post-doxycycline treatment, leading to questions regarding the survival of cells with high CIN (Figure 5D).To examine the relationship between the CIN level and cell death, we stained the cells with Annexin V and propidium iodide (PI), and sorted the "dying cells" (Annexin V + /PI − ), in which nuclear and chromosomal DNA fragmentation had not occurred and the chromosome could still be observed, as well as "living cells" (Annexin V − /PI − ) (Figure 5E).Whereas there was no significant difference between the micronucleus rate of living and dying control cells, the micronucleus rate, as well as the  C) GO B) and Reactome C) enrichments of differentially expressed genes in YY2-overxpressed HCT116 cells.D) Overlapping genes upregulated by YY2 based on RNA-seq, predicted YY2 target genes identified by ChIP-seq, and QuickGO screening.E) BUB3 protein level in YY2-overexpressed HCT116 and HCT116 YY2KO cells, as determined using western blotting.F-H) Schematic diagram (F) as well as relative luciferase activities of BUB3 reporter vectors in HCT116 YY2KO cells (G) and YY2-overexpressed HCT116 cells (H).I) Binding capacity of YY2 to the predicted region in the BUB3 promoter, as determined using ChIP assay.Location of the primer pair used for PCR are shown.J-L) Schematic diagram (J) as well as relative luciferase activities of BUB3-Luc mut in HCT116 YY2KO cells (K) and YY2-overexpressed HCT116 cells (L).M,N) Schematic diagram of YY2 mutants overexpressing vectors (M) and relative luciferase activity of BUB3-Luc-1 in HCT116 cells overexpressing indicated YY2 mutants (N).Cells transfected with pcCon or wild-type HCT116 cells were used as controls.-actin was used as western blotting loading control.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.pcCon: pcEF9-Puro; **P < 0.01; NS: not significant.number of cells with more than one micronucleus in dying YY2overexpressed HCT116 cells, was significantly higher than that in living YY2-overexpressed cells (Figure 5F).Similarly, whereas there was no significant difference in the percentage of polyploid cells between living and dying control cells (3.40% and 3.78%, respectively), the percentage of polyploid cells in dying YY2overexpressed HCT116 cells was significantly higher than that in the corresponding living cells (34.29% and 10.16%, respectively; Figure 5G).Furthermore, while there were only few dying cells in nontreated HCT116 YY2KO cells, we confirmed that the micronucleus rate and percentage of polyploidy cells in those cells were significantly higher than in living HCT116 YY2KO cells (Figure 5H-J).Moreover, while knocking out YY2 alone did not induce cell death and even benefits tumor cell proliferation, treatment with reversine, which could further weaken SAC activity as indicated by the shortened mitotic time (Figure S9C,D and Videos S10, Supporting Information), increased CIN as well as cell death in HCT116 YY2KO cells (Figure S9E,F, Supporting Information).Hence, these results further confirmed the association between cell death with high CIN.It is noteworthy that while excessive CIN, whether due to SAC hyperactivation or due to its hypoactivity, eventually leads to cell death, intrinsic characteristics of the cells, such as different gene expression profiles, might also contribute to the capacity of the cells to tolerate CIN stress, and eventually to their different outcomes such as in cell proliferation. [42,43]Indeed, we observed different expression profiles of genes related with DNA damage response and negative regulation of apoptosis in YY2-overexpressed and YY2knocked out HCT116 cells (Figure S9G, Supporting Information).Nevertheless, while further investigations are needed to systematically analyze these differences, our results clearly suggest that cells with excessive CIN induced by either YY2-mediated SAC hyperactivation or YY2 knockout are more prone to death.
We further noticed that while the level of CIN in the living YY2-overexpressed HCT116 cells was significantly lower than in the dying YY2-overexpressed HCT116 cells, it was slightly higher than in living control cells, as indicated by the slightly increased micronucleus rate and percentage of polyploid cells (Figure 5F,G, respectively).Furthermore, as described above, moderate CIN level is correlated with poor prognosis (Figure 5A), thus raising the question regarding the characteristics of these moderate CIN cells.To examine their characteristics, we reversed YY2 overexpression by withdrawing doxycycline after sorting the living cells to avoid continuous SAC hyperactiva-tion (TetOn-YY2 Trans; Figure S10A,B, Supporting Information).Time-dependent analysis showed that slight increase in micronucleus rate was maintained even at 96 h after doxycycline withdrawal (Figure S10C, Supporting Information), confirming that CIN is heritable and can be passed to the progeny cells as reported previously. [44]Analysis of other CIN indicators 2 days after doxycycline withdrawal further confirmed this result, as the slightly increased CIN level was maintained in living TetOn-YY2 Trans cells (Figure 6A-E).However, unlike the cells continuously overexpressing YY2 (TetOn-YY2 Conti), whose viability and colonyformation potential decreased and cell death rate increased, the viability, colony-formation potential, and cell death rate of TetOn-YY2 Trans cells recovered to the levels similar to those of control despite that they have a certain level of CIN (Figure 6F,G; Figure S10D, Supporting Information).Meanwhile, while continuous YY2 overexpression suppressed the viability and colonyformation potential while increasing cell death rate (TetOn-YY2 Conti), these characteristics recovered to the levels similar to those of control in TetOn-YY2 Trans cells (Figure 6F,G; Figure S10D, Supporting Information).However, under DNA-damage stress induced by oxaliplatin, TetOn-YY2 Trans cells demonstrated robustly higher viability and colony-formation potential (Figure 6H,I).Moreover, the cell death rate of TetOn-YY2 Trans cells was significantly lower than HCT116 cells, which were CINnegative (Figure 6J), suggesting that CIN level before oxaliplatin treatment contribute to the observed differences in viability between cells with continuous and transient YY2 overexpression.Meanwhile, suppressing CIN in TetOn-YY2 Conti cells using reversine decreased cell death and improved cell viability (Figure S10E-G, Supporting Information), indicating that reducing excessive CIN level by inhibiting SAC in SAC-hyperactivated cells can enhance tumor cell drug resistance.Together, these data indicated the correlation between moderate CIN and tumor cell drug resistance.
Residual cells, which survived the anti-tumor drug treatment and undetectable by standard morphological examination during the remission phase between the cycles of chemotherapy, has attracted attention as one of the crucial factors of tumor drug resistance and tumor recurrence, and thus as a hurdle for antitumor therapy. [45]The fact that living cells with YY2-induced SAC hyperactivation possessed moderate CIN suggests the possibility that moderate CIN is also involved in drug resistance in residual tumor cells that survived treatment with DNA damageinducing agents.To test this possibility, we obtained residual tumor cells from CRC cells infected with TetOn-YY2 lentivirus by sorting living cells that survived oxaliplatin treatment, treated them with or without doxycycline to induce YY2 overexpression, and examined their viability under oxaliplatin treatment (Figure 6K).Micronucleus assay revealed that without induction of YY2 overexpression, residual cells that survived oxaliplatin treatment had moderate CIN level (Figure 6L, middle row); furthermore, these cells demonstrated improved DNA-damage stress-resistance compared to that in control cells (Figure 6M).Meanwhile, further induction of CIN by activating YY2 overexpression in these residual cells induced excessive CIN (Figure 6L, right row), leading to better drug sensitivity (Figure 6M).
Together, our results demonstrated that YY2/SAC hyperactivation induces CIN in tumor cells, leading to the mortality of cells with excessive CIN.Moreover, our results suggest that moderate CIN, as observed in residual tumor cells surviving SAC hyperactivation or DNA damage-inducing agent treatment, is crucial for drug resistance in these cells; while increasing CIN in these cells could improve their drug sensitivity.Hence, our findings highlight the crucial role of CIN levels in determining tumor cell survival and drug resistance.

YY2-Induced Excessive CIN Sensitizes Tumor Cells to DNA Damage-Inducing Agent
Given that YY2 overexpression induces excessive CIN, we examined its synergistic effect with oxaliplatin and paclitaxel, which are DNA damage-inducing agents that can induce CIN. [5,46,47]YY2 overexpression significantly decreased the IC 50 of oxaliplatin; in contrast, YY2 knockout robustly increased it (Figure 7A,B).Similarly, YY2 overexpression decreased the IC 50 of paclitaxel (Figure S11A, Supporting Information).Combinatorial index (CI) analysis using a previously described method [48,49] showed a synergistic effect between YY2 overexpression and oxaliplatin treatment (Figure 7C), as well as between YY2 overexpression and paclitaxel treatment (Figure S11B, Supporting Information).The results of the EdU incorporation assay (Figure S11C,D, Supporting Information) and cell death rate (Figure 7D) showed that YY2 overexpression further enhanced the suppressive effect of oxaliplatin on HCT116 cell proliferation potential while further promoting its effect on inducing cell death.
We then assessed the possibility of combining excessive CIN induced by YY2 overexpression with oxaliplatin for anti-tumor therapeutic strategy in vivo.To this end, we established a YY2overexpressed HCT116 stable cell line using a lentivirus and performed xenograft experiments (Figure S11E, Supporting Information).Whereas the volume of xenografted tumors formed by control cells increased 16-fold within 4 weeks, YY2 overexpression or oxaliplatin treatment alone suppressed this increase to approximately eight-fold.The suppressive effect was further en-hanced by combining YY2 overexpression and oxaliplatin treatment, which suppressed the increase in tumor volume to only two-fold (Figure 7E,F).The tumor morphology further confirmed this tendency (Figure 7E).Furthermore, doubling time analysis showed that combining YY2 overexpression and oxaliplatin treatment resulted in more than 9 days of growth delay compared to that with YY2 overexpression or oxaliplatin treatment alone, thereby enhancing the therapeutic effect by approximately four-fold (Figure 7G).Western blotting and immunohistochemistry (IHC) staining results showed that BUB3 expression was increased in tumors formed by YY2-overexpressed cells, whereas oxaliplatin did not affect YY2 and BUB3 expression (Figure 7H; Figure S11F, Supporting Information).Furthermore, IHC staining using H2AX showed that DNA damage in tumor lesions, which was increased by YY2 overexpression or oxaliplatin treatment alone, was further enhanced by combined oxaliplatin treatment and YY2 overexpression (Figure S11F, Supporting Information).
Finally, we examined CIN levels in the tumor lesions.Compared to that in the controls, YY2 overexpression or oxaliplatin treatment increased the chromosome bridge frequency, whereas in oxaliplatin-treated YY2-overexpressed HCT116 cells, this was further increased (Figure 7I).Collectively, these results demonstrated that YY2 overexpression sensitizes tumor cells to DNA damage-inducing agent by increasing CIN, suggesting that this combination might be a potential anti-tumor therapeutic strategy.
Together, our results demonstrated that YY2 is a novel SAC modulator that activates BUB3 transcription, thereby influencing SAC activity.This subsequently determines tumor cell survival and drug resistance by regulating the levels of CIN.

Discussion
Impaired SAC is the main cause of chromosome segregation errors in mitosis, leading to numerical and structural chromosome changes in tumor cells. [12,13]Mouse models have shown that impaired SAC promotes aneuploidy in vivo. [19]Moreover, defective SAC activity is closely related to diseases such as tumors and mosaic variegated aneuploidy, a rare disorder with a high aneuploidy rate and increased tumor incidence. [50]On the other hand, the role of SAC hyperactivation in tumorigenesis is still poorly understood, with conflicting reports on its pro-or antitumor effects. [16,17,39]Nevertheless, mutations in SAC genes are rare in human tumors, indicating that they are not major contributors to the mitotic errors observed in tumor cells. [18]In this study, we found that the expression of YY2, a transcription factor highly conserved in placental mammals, [51,52] oscillates during the cell cycle and peaks during the M phase.Furthermore, we identified YY2 as a novel SAC modulator that directly activates the transcriptional activity of BUB3, a component of the SAC complex.We revealed that YY2 downregulation weakens SAC activity, thereby promoting tumorigenesis by accelerating sister chromatid segregation and mitotic exit.Meanwhile, YY2 overexpression hyperactivates SAC, leading to prolonged mitotic time, decreased proliferation potential, and increased tumor cell death, thus suppressing tumorigenesis.It is noteworthy that even when YY2-knocked out cells were treated with nocodazole, they still retained some level of SAC.This suggests that while YY2 downregulation can weaken SAC activity, it does not abolish it, further underscoring the role of YY2 as a modulator, rather than a component of the SAC.[55] While previous studies have revealed that YY2 can trigger ferroptosis, ultraviolet damage response, and p53-mediated cell cycle arrest, [29,31,56] studies regarding its physiological and pathological functions are still very limited, and the mechanisms underlying its tumor suppressive effect have not been completely elucidated.Hence, our findings provide a new perspective regarding a novel function of YY2 in regulating SAC activity and subsequently, tumor cell mitotic regulation.
CIN is a hallmark of cancer and can drive tumorigenesis.[11] A similar phenomenon was observed in bacteria and viruses, where higher genomic instability benefits the population in stressful environments through the development of mutations that provide a selective growth advantage; however, cells with drastic instability never become dominant in a population, as their instability levels lead to deleterious mutations exceeding the viability threshold. [57]In tumor cells, this concept of a "just-right" level of instability was also observed based on mathematical modeling of the evolutionary dynamics of genetically unstable populations, [58][59][60] suggesting that whereas moderate CIN benefits tumorigenesis as it can allow tumor cells with varying genetic alterations to have greater chance of acquiring advantageous characteristics, such as increased proliferation, ex-cessive CIN might induce tumor cell death.Yet, experimental evidence to support this hypothesis and the molecular mechanisms underlying tumor cell CIN are still lacking.
Intriguingly, YY2 overexpression, which hyperactivates the SAC and triggers tumor suppressive effects, also induces CIN.Indeed, previous studies have revealed that SAC hyperactivation, for example by overexpression of another SAC component, MAD2, or by knocking down p31, a component of SAC silencing, could also induce CIN and cell death. [17,39,61,62]Here we revealed that in the YY2-induced SAC-hyperactivated cell population, dying cells had significantly higher CIN than living cells, thus provides evidence that high CIN induces cell death and is thus deleterious for tumor cells.Meanwhile, although significantly lower than that in dying cells, living cells from this population also exhibited a certain degree of CIN and improved DNA-damage resistance compared to those in control cells, suggesting that moderate CIN is beneficial for tumorigenesis.Together with the fact that knocking out YY2 alone is not sufficient for inducing cell death, while further inhibiting SAC activity in YY2-knocked out cells could also lead to excessive CIN and cell death, our findings clearly showed that different levels of CIN is crucial for determining tumor cell survival.Obviously, while excessive CIN will eventually lead to cell death, the effect of YY2 alteration on intrinsic characteristics of the cells, such as their gene expression profiles in responses to DNA damage stress and apoptotic stimuli, also contribute greatly to their capacity in tolerating CIN stress, and subsequently, the threshold of CIN-induced cell death.Hence, while further systematical investigation is needed to reveal the way tumor cells explore their fitness landscape upon CIN induction, our findings provide experimental evidence that conforms to the "just-right" model. [63,64]NA damage-inducing agents such as oxaliplatin, taxol, and radiotherapy, which have been used clinically for treating tumors, can induce mitotic errors and CIN; [5,10,47,65] while SAC activator, such as MK-1775 and ZN-c3, are in clinical trials and are considered as potential anti-tumor drugs. [66]Meanwhile, increasing evidences suggest that a population of tumor cells remain viable after exposure to various anti-tumor drugs.These residual cells display reduced drug sensitivity, thereby providing a reservoir of cells that might seed the growth of drug-resistant recurrent tumor.Therefore, targeting residual cells have attracted attention as a crucial point for anti-tumor therapeutic strategy; however, the mechanisms by which these cells are generated are still poorly understood.Our findings showed that residual tumor cells that survived DNA damage-inducing agents as well as YY2-induced SAC hyperactivation, possess moderate CIN and are more resistant.Hence, while the underlying mechanism needs to be further elucidated, to our knowledge, our results link up for the first time the level of CIN and residual tumor cells, thus providing new perspective regarding the generation of residual tumor cells as well as their drug resistance.Furthermore, although further study is needed, these findings also point to possible complications, such as predisposing normal or benign tumor cells to becoming more tumorigenic, owing to the induction of moderate CIN mediated by DNA damage-based anti-tumor therapies.Moreover, these findings indicate the possibility of applying the pretreatment CIN level as an indicator to identify patients who would benefit from DNA damage-based anti-tumor therapies.
Subsequently, our results also demonstrated that YY2 overexpression-induced SAC hyperactivation, which triggers excessive CIN, significantly sensitizes CRC cells to oxaliplatin and paclitaxel, indicating a synergism between YY2/SAC hyperactivation-induced excessive CIN and DNA damageinducing agents.Combining YY2 overexpression and oxaliplatin treatment significantly delayed tumorigenesis and enhanced the therapeutic effect in a xenograft mouse model, most plausibly by increasing CIN.Therefore, while further study is required to further elucidate whether DNA damage inducing agents, such as platinum-based and taxol-based antitumor drugs, could induce CIN through SAC activation, as well as the involvement of other YY2-regulated pathways in enhancing the therapeutic effect of this combinatorial therapy, our findings suggest a potential anti-tumor combinatory therapeutic strategy based on a DNA damage-inducing agent and YY2-induced excessive CIN, to improve the efficacy of the former, and at the same time, preventing the generation of residual tumor cells surviving DNA damage-inducing agents with moderate CIN, which could trigger drug resistance.
Taken together, we identified YY2 as a novel modulator of SAC activity.Alterations in this activity can induce different degrees of CIN and influence the survival and drug response of tumor cells.This provides experimental evidence explaining the CIN paradox in cancer.Furthermore, our findings have unraveled a mechanism of drug resistance in residual cells surviving anti-tumor therapies such as those using DNA damage-inducing agents and SAC activation.This suggests a novel anti-tumor therapeutic strategy that combines SAC activity regulators and DNA damage-inducing agents.
Cell Lines and Cell Culture: HCT116 (catalog number: TCHu 99) and HEK293T (catalog number: GNHu17) cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, Life Technologies, Grand Island, NY) with 10% FBS (Biological Industries, Beith Haemek, Israel) and 1% penicillin-streptomycin. Cell lines were verified using short-tandem repeat profiling method and were tested periodically for mycoplasma contamination using Mycoplasma Detection Kit-QuickTest (Biotool, Houston, TX).Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instruction.HCT116 YY2KO cell with deletion of nucleotides located in +95 to +151 region (56 bp) of YY2 coding sequence was established using CRISPR/Cas9 method as described previously. [29]For gene knockdown, overexpression, or rescue experiments, cells were seeded in 6-well plates and transfected with 2 μg of indicated shRNA expression vectors and/or overexpression vectors.24 h after transfection, puromycin selection (final concentration: 1 μg mL −1 ) was performed for 36 h to eliminate untransfected cells.For restoring YY2 in YY2-knocked out cells, HCT116 YY2KO cells Figure 7. Excessive CIN sensitizes tumor cells to DNA-damage agents.A,B) Viability of YY2-overexpressed HCT116 cells (A) and HCT116 YY2KO cells (B) treated with indicated concentrations of oxaliplatin for 24 h.IC 50 was calculated using CompuSyn.C) Combination index (CI) between oxaliplatin and YY2 overexpression, as calculated using CompuSyn.D) Cell death rate of oxaliplatin-treated YY2-overexpressed HCT116 cells, as examined using Annexin V/PI staining and flow cytometry.E) Tumor volume and morphological images of xenografted tumors formed by YY2-overexpressed HCT116 cells and treated with oxaliplatin at indicated time-points (n = 6/group).F) Fold-change of xenografted tumor volumes at day 28 compared to those at the starting point of treatment (day 10).G) Enhancement factor of combinatory treatment of oxaliplatin and YY2 overexpression.H) YY2 and BUB3 protein levels in xenografted tumors, as determined using western blotting.I) Chromosome bridge frequency in the tissue sections of the xenografted tumors.Representative images (scale bars: 5 μm) and quantification results (each dot represents chromosome bridge frequency from one slide, with total 100 mitotic-cells/group) are shown.-actin was used as western blotting loading control.Cells transfected with pcCon, wild-type HCT116 cells, or cells infected with empty virus (EV) were used as controls.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.pcCon: pcEF9-Puro; **P < 0.01.were seeded in 6-well plates and transfected with 1 μg of YY2 overexpression vector before puromycin selection as mentioned above.
Lentiviruses for establishing YY2-inducible cell lines and stable cell lines for xenograft experiments were generated by co-transfecting HEK293T cells with 8 μg pLenti-YY2 or pTRIPZ-YY2 vectors, 6 μg pCMVΔR, and 2 μg pCMV-VSVG in a 10 cm dish.Growth medium was changed the following day and lentivirus-containing supernatant was harvested and filtered with a 0.45-μm filter after 48 h.For infection, HCT116 cells were cultured in 6well plates.24 h later, the medium was changed to 1 mL fresh culture medium and 1 mL corresponding lentivirus supernatant.Infected cells were then selected using 1 μg mL −1 puromycin for 7 days.
For oxaliplatin treatment, cells were seeded in 6-well plates and cultured with medium containing indicated concentration of oxaliplatin (Med-ChemExpress, Monmouth Junction, NJ).
Clinical Human Colon Carcinoma Specimen: Human colon carcinoma specimens were obtained from colon carcinoma patients undergoing surgery at Chongqing University Cancer Hospital (Chongqing, China), and stored in Biological Specimen Bank of Chongqing University Cancer Hospital.Patients did not receive chemotherapy, radiotherapy, or other adjuvant therapies prior to the surgery.The specimens were snap-frozen in liquid nitrogen.Prior patient's written informed consents were obtained.The experiments were approved by the Institutional Research Ethics Committee of Chongqing University Cancer Hospital (Permit No. CZLS2021292-A), and conducted in accordance with Declaration of Helsinki.
Animal Experiments: BALB/c-nu/nu mice (Strain number D000521, male, body weight: 18-22 g, 6 weeks-old) were purchased from the Chongqing Medical University (Chongqing, China), randomly divided into 4 groups (n = 7), and injected subcutaneously with 3 × 10 6 indicated stable cell lines.Oxaliplatin (MedChemExpress) was administered intraperitoneally at a dose of 5 mg kg −1 twice a week for 3 weeks.Treatment began on day 10, when the tumor volume reached 100 mm 3 .Tumor size (V) was evaluated by a caliper every 2 days using the following equation: V = a × b 2 /2, where a and b were the major and minor axes of the tumor, respectively.The investigator was blinded to the group allocation and during the assessment.Animal studies were approved by the Institutional Ethics Committee of Chongqing Medical University (Permit No. SYXK-2021-0001), and carried out in the Chongqing Medical University.All animal experiments conformed to the approved Guidelines for the Care and Use of Laboratory Animals of Chongqing Medical University.All efforts were made to minimize suffering.
Preparation of Cells Transiently Overexpressing YY2: YY2-inducible cell line were treated with doxycycline (final concentration: 2 μg mL −1 ) for at 48 h prior to cell sorting.Following cell sorting, the collected cells were cultured in medium without doxycycline for 48 h to turn off YY2 overexpression.
Tumor Growth Delay and Enhancement Factor: Tumor growth delay and enhancement factor was calculated using the following equation: (1): absolute growth delay was calculated by subtracting the doubling time of the tumor in the treated group from that of the EV group; (2): normalized growth delay for YY2 group was calculated by subtracting the absolute growth delay of the YY2 + oxaliplatin group from that of the EV + oxaliplatin group; (3): normalized growth delay for oxaliplatin was calculated by subtracting the absolute growth delay of the YY2 + oxaliplatin group from that of the YY2 group; (4): enhancement factor for YY2 group was calculated by dividing the normalized growth delay for YY2 group (2) with the absolute growth delay of the YY2 group; (5): enhancement factor for oxaliplatin was calculated by dividing the normalized growth delay for oxaliplatin (3) with the absolute growth delay of the EV + oxaliplatin group.
Live-Cell Imaging: For fluorescence imaging of mitotic division, cells were prepared as described above prior to seeding in 3.5-cm glass bottom dish (Wuxi NEST Biotechnology, Wuxi, China; 1 × 10 5 cells per dish).Nuclei was stained using Hoechst 33342 (Solarbio, Beijing, China) 30 min prior to imaging.The culture was maintained at 37 °C under 5% CO 2 in a stage-top incubator (Tokai Hit, Shizuoka, Japan).Images were acquired every 5 min for 6 h with a 20-ms exposure time using inverted fluorescence microscope Olympus IX83 (Olympus).Images were processed using FLU-OVIEW (v.2.3).Mitotic time was quantified as the time from nuclear envelope breakdown (NEBD) until the onset of anaphase.
To study cumulative mitotic exit, nocodazole (final concentration: 100 ng mL −1 ; Beyotime Biotechnology) was added, and the cells were imaged every 10 min for 10 h using Olympus IX83 (Olympus).The number of cells that exited mitosis was quantified and analyzed over time using FLUOVIEW software (Olympus).
RNA Sequencing and Data Analysis: RNA-seq analysis were performed by Shanghai Bio Technology Corporation (Shanghai, China) using Illumina HiSeq 2500 (Illumina, San Diego, CA; three replicates for each group).Sequencing raw reads were pre-processed by filtering out rRNA reads, sequencing adapters, short-fragment reads and other low-quality reads.Tophat v2.1.0was used to map the cleaned reads to human reference genome ensemble GRCh38 (hg38) with two mismatches.After genome mapping, Cufflinks v2.1.1 was run with a reference annotation to generate FPKM values for known gene models.Differentially expressed genes were identified using Cuffdiff.The P-value significance threshold in multiple tests was set by the false discovery rate (FDR).The fold-changes were also estimated according to the FPKM in each sample.To identify DEGs in YY2-overexpressed cells, pairwise comparisons between each transfected sample and each control sample were first performed to identify genes upregulated or downregulated in each YY2-overexpressed sample with an FDR ≤ 0.05 relative to each control.
CIN70 Scoring and Survival Data Analysis: CIN70 scores were calculated as the mean expression of the 70 probe sets matching the 70 genes of the CIN70 signature. [41]The expression level of the 70 genes of CIN70 was obtained from TCGA dataset of CRC patients treated with oxaliplatin (cBioportal; coadread_tcga_pan_can_atlas_2018).All CRC samples were stratified into CIN70 quartiles according to the CIN70 scores.
Metaphase Spread: Cells were prepared as described above and arrested at metaphase by 2 μg mL −1 colchicine (Aladdin, Shanghai, China) treatment for 3 h before being harvested, re-suspended in hypotonic solution (0.075 M KCl) and incubated for 15 min at 37 °C.After cell swelling, cells were fixed using 2 mL of freshly prepared methanol-acetic acid fixative (3:1) and collected by centrifugation.The pellet was re-suspended in 5 mL of 3:1 methanol-acetic acid for 30 min and dropped onto pre-cooled slides.Images of mitotic chromosomes were acquired with fluorescence microscope (Olympus BX53, Olympus, Tokyo, Japan).Chromosome number per cell was quantified.
Karyotype and G-band Analysis: Cells were treated with 2 μg mL −1 colchicine for 1 h and collected processed as described above.After dropped onto pre-cooled slides, the slides were allowed to dry overnight at room temperature, banded with trypsin and stained with 10% Giemsa stain (Biosharp, Shanghai, China).Images were taken using light microscopy with Olympus BX53 (Olympus) and analyzed using SmartType software (Digital Scientific, Cambridge, UK). 10 metaphase spread images were analyzed for each sample.
Cell Cycle Enrichment and DNA Content Analysis: For cell cycle analysis, cells were prepared as described above and synchronized in G 0 phase by FBS starvation for 24 h.Cells were washed two times in phosphatebuffered saline (PBS) and either collected immediately (0 h) or further cultured in medium with 10% FBS for indicated time points.
For DNA content analysis, cells were prepared as described above and subjected to flow cytometry after staining the DNA with propidium iodide (PI) (NeoBiosciences).Percentage of cells with each DNA content (2N, 3N, 4N, 5-8N) were estimated using CytExpert software (Beckman Coulter).For DNA content analysis using sorted cells, cells were transfected or infected with indicated vectors and sorted for living and dying cells as described above before being stained with PI.
Chromosome Bridge and Micronucleus Staining: Cells were prepared as described above and cultured in 3.5-cm confocal dishes (3 × 10 4 cells/dish) for overnight, and synchronized at prometaphase by nocodazole treatment (final concentration: 100 ng mL −1 ; Beyotime Biotechnology) for 6 h.Cells were then fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with PBS containing 0.1% Triton X-100 for 5 min.Nuclei were stained with DAPI (Beyotime Biotechnology) for 15 min.For chromosome bridge observation, images were taken using laser scanning confocal microscopy (Leica Microsystems TCS SP5, Heidelberg, Germany).For micronuclei observation, images were taken using fluorescence microscope (Olympus IX71, Olympus).Micronucleus rate was defined as the ratio of total micronuclei to total cell number.
For chromosome bridge observation in xenografted tumor lesions, fresh xenografted tumor lesions were fixed using 4% paraformaldehyde for overnight, embedded in paraffin, and sectioned at 4 μm thickness using a cryostat.After being dewaxed using xylene and rehydrated, sections were stained with 0.5% hematoxylin-eosin (Sangon Bio, Shanghai, China).After samples were dehydrated and mounted in coverslip, images were taken using light microscope (Olympus BX53, Olympus).
Cell Death Assay: Cells were prepared as described above in Materials and Methods and re-seeded in a 6-well plate (3 × 10 5 cells/well).Cells were stained with Annexin V/PI (NeoBiosciences) according to manufacturer's instruction 24 h after transfection and subjected to flow cytometry.Data was analyzed using CytExpert software.
Cell Viability Assay and Calculation of IC 50 : Cells were prepared as described above, re-seeded into 96-well plates (5 × 10 3 cells/well), and treated with oxaliplatin (final concentration: 1 μM).Preparation and Analysis of Residual Tumor Cells: YY2-inducible cell line was prepared as described above and cultured without doxycycline induction.Cells were treated with oxaliplatin (final concentration: 1 μM) for 24 h prior to sorting for living cells as described above.Oxaliplatin was withdrawn after cell sorting, and living cells were further cultured in medium with or without doxycycline (final concentration: 2 μg mL −1 ) for 48 h.Cells were treated with oxaliplatin (final concentration: 1 μM) for 5 days, and cell viability was analyzed as described previously.
For micronucleus analysis, living cells were further cultured with or without doxycycline (final concentration: 2 μg mL −1 ) for 48 h after sorting.Micronucleus staining was performed as described above in Materials and Methods.
5-ethynyl-2′-deoxyuridine (EdU) Incorporation Assay and Colony-formation Assay: Cells were prepared as described above and then re-seeded in 48well plate (5 × 10 4 cells/well).EdU incorporation and staining were performed using BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime Biotechnology) according to the manufacturer's instruction.Nuclei were stained with Hoechst.Images were taken with fluorescence microscope (Olympus IX71).Quantification of EdU-positive and Hoechstpositive cells were shown as the ratio of EdU-positive cells to Hoechstpositive cells.
For colony-formation assay, 300 cells were cultured in a 6-well plate for 10 days.Cells were then fixed with 4% paraformaldehyde and stained with methylene blue.The colonies were then counted.The investigator was blinded during the assessment.
Dual Luciferase Reporter Assay: Cells were seeded into 24-well plates (8 × 10 4 cells/well).Twenty-four hours later cells were co-transfected with the indicated overexpression vector, reporter vector, and Renilla luciferase expression vector (pRL-SV40, Promega) as the internal control.Luciferase activities were measured using Dual Luciferase Assay System (Promega) 48 h after transfection.Firefly luciferase activities were normalized with the corresponding Renilla luciferase activities.
RNA Extraction and Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted using TRIzol (Invitrogen Life Technology, Carlsbad, CA) according to the manufacturer's instructions.Total RNA (1 μg) was then reverse transcribed into cDNA using a PrimeScript Reagent Kit with gDNA Eraser (Takara Bio).qRT-PCR was performed using SYBR Premix Ex Taq (Takara Bio).The sequences of the primers used were listed in Table S1 (Supporting Information).-actin was used to normalize sample amplifications.
Western Blotting: Total cells were lysed with RIPA lysis buffer supplemented with a protease inhibitor and phosphatase inhibitor cocktail (complete cocktail, Roche Applied Science, Mannheim, Germany).For samples from xenografted tumors, frozen specimens were homogenized with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail to obtain protein extracts.Samples with equal amounts protein were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels before being transferred to a polyvinylidene fluoride membrane with 0.45-μm pore size (Millipore, Billerica, MA).Antibodies used were listed in Table S2 (Supporting Information), and immunoblotting with an anti--actin antibody was conducted to ensure equal protein loading.Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific).
Protein Degradation Assay: For protein degradation assay, cells were synchronized at prometaphase by nocodazole treatment, as described above in Materials and Methods.Mitotic cells were washed two times in PBS and either collected immediately (0 h) or released in drug-free medium.Protein samples were collected at the indicated time points and were subjected to western blotting as described above.Protein half-life was determined by quantifying western blotting results.
Immunohistochemistry and Hematoxylin-Eosin Staining: Fresh xenografted tumor lesions were fixed using 4% paraformaldehyde for overnight, embedded in paraffin, and sectioned at 4 μm thickness using a cryostat.After being dewaxed using xylene and rehydrated, sections were subjected to immunohistochemical staining.Briefly, the tissue sections were incubated with primary antibodies for 1 h.The specimens were then incubated with corresponding second antibodies conjugated with horse-radish peroxidase.Visualization was performed using a DAB Kit (DAKO, Beijing, China) under microscope.The nuclei were then counterstained with hematoxylin (Beyotime Biotechnology), then the sections were dehydrated and mounted with coverslip.Antibodies used were listed in Table S2 (Supporting Information).Images were taken by using Pannoramic Midi (3DHistech, Budapest, Hungary).
Statistical Analysis: -actin was used for normalization in qRT-PCR experiment and in quantifying protein expression levels for calculation of protein half-life.For dual luciferase reporter assay, firefly luciferase activities were normalized with those of Renilla.All values of the experimental results were presented as mean ± SD (n = 3; unless further indicated).Quantification data were analyzed by one-way ANOVA conducted using SPSS Statistics v20.0 (IBM, Chicago, IL).A value of P < 0.05 was considered statistically significant.

Figure 1 .
Figure 1.Identification of novel transcriptional regulators modulating SAC.A) Schematic diagram of screening strategy for potential novel transcriptional regulators modulating SAC.B) Top 17 potential TFs modulating SAC obtained from ChEA analysis.C) mRNA expression levels of potential novel SAC modulators at each cycle phase, as examined using qRT-PCR.D) YY2, cyclin D, and cyclin B protein expression levels in HCT116 cells at indicated timepoints after serum starvation release, as determined by western blotting.E-F) Mitotic time of YY2-overexpressed HCT116 cells (E) and HCT116 YY2KO cells (F), as determined using time-lapse microscopy.Representative images (scale bars: 20 μm) are shown.G-H) Scatter plots showing the timelength from nuclear envelope breakdown (NEBD) to anaphase of YY2-overexpressed HCT116 cells (G) and HCT116 YY2KO cells (H) (total n = 60, pooled from three independent experiments).I,J) Time-lapse analysis of duration of mitosis in YY2-overexpressed HCT116 cells (I) and HCT116 YY2KO cells (J) arrested with nocodazole (final concentration: 100 ng mL −1 ; total n = 150, pooled from three independent experiments).Cells transfected with pcCon, wild-type HCT116, or unsynchronized cells were used as controls.-actin was used for qRT-PCR normalization and as western blotting loading control.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way analysis of variance (ANOVA).pcCon: pcEF9-Puro; Unsync: unsynchronized; **P < 0.01; NS: not significant; ND: not detected.

Figure 2 .
Figure 2. YY2 suppresses tumorigenesis by activating SAC activity.A,B) Cell death rate of YY2-overexpressed HCT116 (A) and HCT116 YY2KO cells (B), as examined using Annexin V/PI staining and flow cytometry.C,D) Mitotic time of YY2-overexpressed, reversine-treated HCT116 cells, as determined using time-lapse microscopy.Representative images (C; scale bars: 20 μm) and scatter plot showing the time-length from NEBD to anaphase (D; total n = 60, pooled from three independent experiments) are shown.E) Proliferation potential of YY2-overexpressed, reversine-treated HCT116 cells, as determined using EdU-incorporation assay.Representative images (left; scale bars: 200 μm) and ratio of proliferative cells (right; each dot represents the mean value of three technical replicates) are shown.F) Cell death rate of YY2-overexpressed, reversine-treated HCT116 cells, as examined using Annexin V/PI staining.Cells transfected with pcCon or wild-type HCT116 cells were used as controls.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.pcCon: pcEF9-Puro; Rev: reversine (final concentration: 0.2 μM); **P < 0.01; NS: not significant.

Figure 3 .
Figure 3. YY2 directly regulates BUB3 transcription.A) Volcano plot of log2 fold-change versus adjusted P value for gene expression changes (fold-change > 1.01-fold; P value < 0.05) in YY2-overexpressed HCT116 cells, as analyzed by RNA-seq.B-C) GO B) and Reactome C) enrichments of differentially expressed genes in YY2-overxpressed HCT116 cells.D) Overlapping genes upregulated by YY2 based on RNA-seq, predicted YY2 target genes identified by ChIP-seq, and QuickGO screening.E) BUB3 protein level in YY2-overexpressed HCT116 and HCT116 YY2KO cells, as determined using western blotting.F-H) Schematic diagram (F) as well as relative luciferase activities of BUB3 reporter vectors in HCT116 YY2KO cells (G) and YY2-overexpressed HCT116 cells (H).I) Binding capacity of YY2 to the predicted region in the BUB3 promoter, as determined using ChIP assay.Location of the primer pair used for PCR are shown.J-L) Schematic diagram (J) as well as relative luciferase activities of BUB3-Luc mut in HCT116 YY2KO cells (K) and YY2-overexpressed HCT116 cells (L).M,N) Schematic diagram of YY2 mutants overexpressing vectors (M) and relative luciferase activity of BUB3-Luc-1 in HCT116 cells overexpressing indicated YY2 mutants (N).Cells transfected with pcCon or wild-type HCT116 cells were used as controls.-actin was used as western blotting loading control.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.pcCon: pcEF9-Puro; **P < 0.01; NS: not significant.

Figure 4 .
Figure 4.Both YY2 knockout and overexpression induces CIN.A) DNA content in HCT116YY2KO  cells, as analyzed using PI staining and flow cytometry.B) Average nuclei size of DAPI-stained HCT116 YY2KO cells (each dot represents average nuclei size from one independent experiment, with total 100 cells/group).C) Chromosome number per cell in HCT116 YY2KO cells, as analyzed using metaphase spread.Representative images (scale bars: 10 μm) and percentage of cells with indicated chromosome number (total cells counted: 50 cells/group, pooled from three independent experiments) are shown.D) Single karyotype analysis of HCT116 YY2KO cells.Representative images and percentage of cells with indicated chromosome number (total cells counted: 10 cells/group, pooled from three independent experiments) are shown.E) Chromosome bridge frequency in HCT116 YY2KO cells.Representative images (scale bars: 5 μm) and quantification results (each dot represents chromosome bridge frequency from one independent experiment, with total 100 mitotic-cells/group) are shown.F) Micronucleus rate in HCT116 YY2KO cells.Representative images of micronuclei (indicated by arrowheads; scale bars: 20 μm) and micronucleus rate (ratio of micronuclei number to total cell number; each dot represents micronucleus rate/slide with > 100 cells/slides; four technical replicates from three independent experiments) are shown.G-L) DNA content analysis (G), average nuclei size (H), chromosome number per cell (I), single karyotype analysis (J), chromosome bridge frequency (K), and micronucleus rate (L) in YY2-overexpressed HCT116 cells.Cells transfected with pcCon or wild-type HCT116 cells were used as controls.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.pcCon: pcEF9-Puro; *P < 0.05; **P < 0.01.

Figure 5 .
Figure 5. CIN level determines tumor cell survival.A) Kaplan-Meier relapse-free survival curves of 108 oxaliplatin-treated CRC patients stratified by CIN70 score quartile.B,C) Cell death rate of HCT116 cells after YY2 induction by doxycycline (B) and after reversine treatment (final concentration: 0.2 μM) (C), as examined using Annexin V/PI staining.D) Micronucleus rate in HCT116 cells after YY2 induction by doxycycline.Representative images of micronuclei (indicated by arrowheads; scale bars: 20 μm) and micronucleus rate (ratio of micronuclei number to total cell number; each dot represents micronucleus rate/slide with > 100 cells/slides; four technical replicates from three independent experiments) are shown.E-G) Sorting (E), micronucleus rate (F), and DNA content (G) of living (L) and dying (D) YY2-overexpressed HCT116 cells.H-J) Sorting (H), micronucleus rate (I), and DNA content (J) of living L) and dying (D) HCT116 YY2KO cells.Cells transfected with pcCon, wild-type HCT116 cells, or HCT116 cells infected with lentivirus generated using pTRIPZ-control (Con) were used as controls.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.Scale bars: 20 μm; pcCon: pcEF9-Puro; Dox: doxycycline (final concentration: 2 μg mL −1 ); **P < 0.01; NS: not significant.

Figure 6 .
Figure 6.Residual tumor cells have moderate CIN and increased drug resistance.A) DNA content in HCT116 cells transiently overexpressing YY2 (TetOn-YY2 Trans cells) 48 h after sorting, as analyzed using PI staining and flow cytometry.B) Average nuclei size of TetOn-YY2 Trans cells 48 h after sorting (each dot represents average nuclei size from one independent experiment, with total 100 cells/group).C) Chromosome number per cell in TetOn-YY2 Trans cells 48 h after sorting, as analyzed using metaphase spread.Representative images (scale bars: 10 μm) and percentage of cells with indicated chromosome number (total cells counted: 50 cells group −1 , pooled from three independent experiments) are shown.D) Chromosome bridge frequency in TetOn-YY2 Trans cells 48 h after sorting.Representative images (scale bars: 5 μm) and quantification results (each dot represents chromosome bridge frequency from one independent experiment, with total 100 mitotic-cells/group) are shown.E) Micronucleus rate of TetOn-YY2 Trans cells 48 h after sorting.Representative images of micronuclei (indicated by arrowheads; scale bars: 20 μm) and micronucleus rate (ratio of micronuclei number to total cell number; each dot represents micronucleus rate/slide with > 100 cells/slides; four technical replicates from three independent experiments) are shown.F,G) Cell death rate at indicated time-points (F) and cell viability at 4 days after sorting (G) of TetOn-YY2 Trans cells.H-J)Viability (H), colony-formation potential (I; each dot represents the mean value of three technical replicates), and cell death rate (J) of TetOn-YY2 Trans cells at indicated times, 10 days, and 5 days after oxaliplatin treatment, respectively.K-M) Schematic diagram (K), micronucleus rate (L), and viability after oxaliplatin treatment (M) of residual HCT116 cells infected with TetOn-YY2 lentivirus.Cells infected with control lentivirus generated from pTRIPZcontrol (Con) were used as controls.TetOn-YY2 Trans and Conti: cells infected with TetOn-YY2 lentivirus and treated with doxycycline only before sorting or continuously, respectively.Pre-sort and post-sort: pre-sorting and post-sorting.Quantification data are shown as mean ± SD.All data were obtained from three independent experiments.P values were calculated by one-way ANOVA.Dox: doxycycline (final concentration: 2 μg mL −1 ); **P < 0.01; NS: not significant.